Rifabutin treatment methods, uses, and compositions

ABSTRACT

The invention provides formulations containing highly concentrated solutions of rifabutin and methods of making such formulations. The invention also provides methods of using such formulations to treat a bacterial infection in a subject.

RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. ProvisionalPatent Application Ser. Nos. 62/902,019, filed Sep. 18, 2019,62/899,257, filed Sep. 12, 2019, 62/941,160, filed Nov. 27, 2019, and62/977,659, filed Feb. 17, 2020, the content of each of which isincorporated herein in its entirety

FIELD OF THE INVENTION

The invention generally related to formulations containing theantibiotic rifabutin, methods of the making such formulations, andmethods of using such formulations in the treatment of bacterialinfections.

BACKGROUND

Millions of people die each year from bacterial infections, and thenumbers are increasing due to the spread of antibiotic-resistantbacterial strains. For example, according to official estimates, theannual number of deaths due to infections from antibiotic-resistantbacteria in the United States, European Union, and India alone is over100,000, and some experts believe that official tallies are vastunderestimates because the full impact of antibiotic resistance is stillunknown. Unfortunately, the pipeline for development of new antibioticsin recent decades has slowed to a trickle, and many existing antibioticsare beset with problems that limit their effectiveness.

One existing antibiotic that has failed to reach its full therapeuticpotential is rifabutin, also known as LM427 and Mycobutin®. Althoughrifabutin is active against a broad spectrum of bacteria, its poorwater-solubility makes the antibiotic difficult to deliver at doseseffective for treating infections other than those reported in theMycobutin® label and to effectively prevent the development ofresistance. Based on studies on rifabutin and related antibiotics of thesame class, it is believed that high levels of free rifabutin arenecessary both for microbial killing and to prevent development ofresistance. Consequently, the therapeutic utility of rifabutin ishampered by technical problems, and millions of people continue tosuffer from bacterial infections due to the lack of adequate treatmentoptions.

SUMMARY

The invention provides pharmaceutical formulations that contain highconcentrations of rifabutin. Preferred compositions of the inventioncomprise a rifabutin powder formulated in water, a solvent and an acid.The inventive combination allows dissolution of rifabutin at highconcentration. The formulated solution may be diluted withoutrestriction in order to render a composition suitable for a desiredroute of administration.

Formulations of the invention allow delivery of effective amounts ofrifabutin by routes of administration that are not possible with priorrifabutin-containing compositions. For example, formulations of theinvention enable rifabutin to be provided parenterally, includingintravenously or by inhalation. In addition, formulations of theinvention obviate the need to lyophilize rifabutin prior toreconstituting it for administration, a rather costly process.

In another aspect of the invention, rifabutin is prepared as a powderwith a shelf-life equivalent to that of the rifabutin activepharmaceutical ingredient (API). As detailed below, rifabutinformulations of the invention preferably comprise rifabutin powderdissolved in an organic solvent.

According to the invention, highly concentrated rifabutin formulationsare rapidly obtained from any rifabutin API and used as such or furtherdiluted ad libitum with sterile water or pharmaceutically-acceptablesolutions. Formulations of the invention are useful for treating avariety of conditions caused by or associated with bacterial infections,such as, but not limited to, bacteremia, meningitis,Ventilator-Associated Bacterial Pneumonia (VABP), Hospital-AcquiredBacterial Pneumonia (HABP) and Periprosthetic Joint Infections (PJI).

In one aspect, the invention provides non-oral formulations of rifabutinmanufactured by preparing a solution in the presence of an acid suitableto promote dissolution of rifabutin. The solution preferably includes asolvent and water in a ratio appropriate for the intended use of theformulation.

A formulation of the invention is suitable for any non-oral route ofadministration. The formulation is suitable for parenteral, intravenous,intraarterial administration, or pulmonary delivery. The formulation isalso suitable for administration by inhalation or by injection.

Formulations of the invention are reconstituted solutions that may needto be diluted prior to non-oral administration. A formulation of theinvention includes solvent and water in a defined ratio. The ratio maybe a v/v ratio. The solution may include the solvent and distilled waterin a ratio of from about 9:1 to about 1:9, from about 9:1 to about 1:4,from about 9:1 to about 1:2, from about 9:1 to about 1:1, from about 4:1to about 1:9, from about 4:1 to about 1:4, from about 4:1 to about 1:2,from about 4:1 to about 1:1, from about 2:1 to about 1:9, from about 2:1to about 1:4, from about 2:1 to about 1:2, or from about 2:1 to about1:1. The solution may include the solvent and distilled water in ratioof about 9:1, about 4:1, about 2:1, about 1:1, about 1:2, about 1:4, orabout 1:9.

The solvent may be polyoxyethylene sorbitan monooleate (Tween 80),sorbitan monooleate polyoxyethylene sorbitan monolaurate (Tween 20),polyethylene glycol (PEG), propylene glycol, N-methyl-2-pyrrolidone(NMP), glycerin, ethanol, dimethylacetamide (DMA), diethylene glycolmonoethyl ether (transcutol HP), or dimethyl isosorbide (DMI).

The acid may be hydrochloric, methanesulfonic, phosphoric, L-tartaric,D-glucuronic, L-malic, D-gluconic, L-lactic, acetic, or L-aspartic acid.

A reconstituted solution of the invention preferably contains about 250mg/ml (1:1 solvent/water) or about 166.7 mg/ml. (1:2 solvent/water),however concentrations of the reconstituted solution may be as high asabout 300 mg/ml. In certain embodiments, a more dilute solution isrequired and that is obtained by adding more water to the solvent. Forexample, rifabutin in a 1:4 solvent/water ratio will result in asolution of about 50 mg/ml. However, such a dilution will takeadditional time to dissolve rifabutin powder. Alternatively, rifabutincan be dissolved in a solvent and reconstituted solutions are thenobtained without further modification. In general, for an IV solution,the desire is to keep the rifabutin/solvent ratio as low as possible.Appropriate ranges are provided herein.

Formulations of the invention are effective for treatment of a bacterialinfection. The infection may include one or more of A. baumannii, C.jejuni, C. trachomatis, H. ducreyi, H. influenzae, H. pylori, M.chelonae, M. kansasii, M. leprae, M. tuberculosis, Mycobacterium avium,Mycobacterium intracellulare, N. gonorrhoeae, N. meningitidis,staphylococci, streptococci (e.g., group A streptococci), and T. gondiior any other pathogen that is susceptible to rifabutin.

The amount of acid relative to rifabutin may be between 1 and 3 molarequivalents or between 1 and 2 molar equivalents. The amount of acidrelative to rifabutin may be 1 molar equivalent.

The w/v ratio of rifabutin to solvent may be from about 4:1 to about1:4, from about 2:1 to about 1:3, or from about 1:1 to about 1:2. Thew/v ratio of rifabutin to solvent may be about 4:1, about 3:1, about2:1, about 1:1, about 1:2, about 1:3, or about 1:4.

In another aspect, the invention provides methods of preparing anon-oral formulation of rifabutin by preparing a solution including asolvent and distilled water, adding an acid to the solution, andintroducing this solution to rifabutin powder, causing the rifabutin todissolve in the solution.

In another aspect, the invention provides methods of preparing anon-oral formulation of rifabutin by preparing a solution of rifabutinin solvent and a solution of the acid in water and then mixing the twosolutions.

The formulation may have any of the properties described above inrelation to formulations. The acid and solvent may be any of thosedescribed above. The water and solvent may be combined at any ratiodescribed above.

Methods of the invention include diluting a formulated rifabutinsolution without restriction in order to render a composition suitablefor a desired route of administration into a pharmaceutically acceptablediluent such as, but not limited to, sterile water, sodium chloride(i.e., saline) solution, dextrose water, Ringer lactate solution.

The step of dissolving the rifabutin may include swirling, stirring, oragitation of the solution. The step of dissolving the rifabutin may beperformed for a defined period. The step of dissolving the rifabutin maybe performed for about 5 minutes, about 10 minutes, about 15 minutes,about 20 minutes, about 30 minutes, about 45 minutes, or about 60minutes.

The rifabutin may be provided as a solid powder.

The rifabutin may be provided as a solution in solvent.

In another aspect, the invention provides methods of treating abacterial infection in a subject by administering a therapeuticallyeffective amount of a non-oral formulation of rifabutin.

The bacterial infection may include one or more of A. baumannii, C.jejuni, C. trachomatis, H. ducreyi, H. influenza, H. pylori, M.chelonae, M. kansasii, M. leprae, M tuberculosis Mycobacterium avium,Mycobacterium intracellulare, N. gonorrhoeae, N. meningitidis,staphylococci, streptococci (e.g., group A streptococci), and T. gondiior any other pathogen that is susceptible to rifabutin.

The formulation may have any of the properties described above inrelation to formulations.

The formulation may be provided parenterally, intravenously, or byinhalation.

Aspects of the disclosure provide a use of rifabutin, an acid, asolvent, and a diluent for making a medicament for treating a bacterialinfection.

In certain embodiments, the diluent is water.

In certain embodiments, the w/v ratio of rifabutin to solvent may beabout 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, orabout 1:4.

In certain embodiments, the w/v ratio of rifabutin to solvent is about1:2.

In certain embodiments, the solvent is polyoxyethylene sorbitanmonooleate (Tween 80), sorbitan monooleate polyoxyethylene sorbitanmonolaurate (Tween 20), polyethylene glycol (PEG), propylene glycol,N-methyl-2-pyrrolidone (NMP), glycerin, ethanol, dimethylacetamide(DMA), diethylene glycol monoethyl ether (transcutol HP), or dimethylisosorbide (DMI).

In certain embodiments, the solvent is DMI or transcutol HP.

In certain embodiments, the solvent and water are present in a ratio offrom about 9:1 to about 1:9, from about 9:1 to about 1:4, from about 9:1to about 1:2, from about 9:1 to about 1:1, from about 4:1 to about 1:9,from about 4:1 to about 1:4, from about 4:1 to about 1:2, from about 4:1to about 1:1, from about 2:1 to about 1:9, from about 2:1 to about 1:4,from about 2:1 to about 1:2, or from about 2:1 to about 1:1.

In certain embodiments, the solvent and water are present in a ratio offrom about 1:1 to about 1:2.

In certain embodiments, the acid is hydrochloric, methanesulfonic,phosphoric, L-tartaric, D-glucuronic, L-malic, D-gluconic, L-lactic,acetic, or L-aspartic acid.

In certain embodiments, the acid may be D-glucoronic acid.

In certain embodiments, the acid may be acetic acid.

In certain embodiments, the amount of acid relative to rifabutin isbetween 1 and 3 molar equivalents or between 1 and 2 molar equivalents.

In certain embodiments, the amount of acid relative to rifabutin may is1 molar equivalent.

In certain embodiments, the rifabutin to acid molar ratio is about 1:1.

In certain embodiments, the w/v ratio of rifabutin to solvent may befrom about 4:1 to about 1:4, from about 2:1 to about 1:3, or from about1:1 to about 1:2.

In certain embodiments, the bacterial infection is A. baumannii, C.jejuni, C. trachomatis, H. ducreyi, H. influenza, H. pylori, M.chelonae, M. kansasii, M. leprae, M tuberculosis Mycobacterium avium,Mycobacterium intracellulare, N. gonorrhoeae, N. meningitidis,staphylococci, streptococci (e.g., group A streptococci), or T. gondii.

In another aspect, the invention provides formulations comprisingrifabutin, an acid, water, and a solvent suitable to promote dissolutionof the rifabutin.

The formulations may contain any ratio of rifabutin to solvent, anyratio of solvent to water, or any ratio of rifabutin to acid describedabove.

The formulations may contain any solvent or any acid described above.

The formulation may contain any concentration of rifabutin, such asabout 250 mg/ml, about 200 mg/ml, about 150 mg/ml, about 100 mg/ml,about 50 mg/ml, about 20 mg/ml, about 10 mg/ml, about 5 mg/ml, about 2.5mg/ml, about 1 mg/ml, at least about 250 mg/ml, at least about 200mg/ml, at least about 150 mg/ml, at least about 100 mg/ml, at leastabout 50 mg/ml, at least about 20 mg/ml, at least about 10 mg/ml, atleast about 5 mg/ml, at least about 2.5 mg/ml, at least about 1 mg/ml,from about 1 mg/ml to about 250 mg/ml, from about 2.5 mg/ml to about 250mg/ml, from about 5 mg/ml to about 250 mg/ml, from about 10 mg/ml toabout 250 mg/ml, from about 20 mg/ml to about 250 mg/ml, from about 50mg/ml to about 250 mg/ml, from about 100 mg/ml to about 250 mg/ml, fromabout 1 mg/ml to about 200 mg/ml, from about 2.5 mg/ml to about 200mg/ml, from about 5 mg/ml to about 200 mg/ml, from about 10 mg/ml toabout 200 mg/ml, from about 20 mg/ml to about 200 mg/ml, from about 50mg/ml to about 200 mg/ml, or from about 100 mg/ml to about 200 mg/ml.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . is a schematic illustration of a method for preparing aninjectable rifabutin solution or rifabutin for inhalation according toembodiments of the invention.

FIG. 2 is a schematic illustration of methods of analysis of rifabutinformulations.

FIG. 3 is a graph showing rifabutin solubility in formulations.

FIG. 4 is a graph showing rifabutin solubility in formulations.

DETAILED DESCRIPTION Rifabutin and Challenges of Administering Rifabutin

The invention provides compositions and methods for the preparation ofsolutions containing rifabutin suitable for parenteral or inhalatoryadministration. Importantly, the invention enables intravenousadministration of rifabutin at high doses. The intravenous formulationsof rifabutin permit delivery of the compound with much higher efficiencyand efficacy than that can be achieved with prior oral rifabutinformulations. In particular the invention discloses the use ofwater/solvent mixtures in the presence of an acid as a pharmaceuticallyacceptable solution for reconstitution of rifabutin powder for a rapidpreparation of a stable highly-concentrated reconstituted solution,which, in turn, can be diluted without restriction with additional waterfor injection or with pharmaceutically acceptable diluents in order torender a composition suitable for a desired route of administration.

Formulations of the invention are related to the requirement that highconcentrations of rifabutin at the site of infection are optimal forreaching the appropriate Pharmacokinetics (PK) parameters, such as AreasUnder the Curve (AUC) and C_(max), which are needed for highest clinicalefficacy and prevention of resistance in the treatment of bacterialinfections against which rifabutin is active.

Rifabutin is a dark red-violet powder, has a molecular formula ofC₄₆H₆₂NO₁₁, a molecular weight of 847.02 and the following structure:

Rifabutin has a broad spectrum of antimicrobial activity. It isconsiderably more active than rifampin against MAC, M. tuberculosis, andM. leprae. It is also active against most atypical mycobacteria,including M. kansasii; M. chelonae, however, is relatively resistant.Rifabutin is also active against staphylococci, group A streptococci, N.gonorrhoeae, N. meningitidis, H. influenza, H. ducreyi, C. jejuni, H.pylori, C. trachomatis, T. gondii and A. baumannii.

In healthy adult volunteers a nominal therapeutic oral dose of 300 mgRifabutin produces a mean C_(max) of 0.375 mg/L which is attained atapproximately 3 hours after oral administration (rifabutin productmonograph). The PK of rifabutin is linear after single administration of300, 450 and 600 mg PO to healthy volunteers with a C_(max) in the rangeof 0.4 to 0.7 mg/L (rifabutin product mongraph). In a study inHIV-infected patients receiving the recommended daily dose of rifabutin(300 mg/day) the plasma concentrations at steady state wereCmax=0.59±0.33 mg/L and the AUC was 8.6±8.2 mg*h/L (Hafner et al, 1998).Since rifabutin is approximately 90% protein bound the free drugconcentrations after oral administration are very low. In healthy adultvolunteers at least 53% of the oral dose is absorbed whereas theabsolute bioavailability assessed in HIV-positive patients in a multipledose study was 20% on day 1 and 12% on day 28.

In closely related rifamycin molecules, such as rifampicin, microbialkilling is linked to the area under the concentration-timecurve-to-minimum-inhibitory-concentration (MIC) ratio (AUC/MIC) whereasthe suppression of resistance was associated with the free peakconcentration (C_(max)-to-MIC ratio (C_(max)/MIC) and not to theduration that the rifampin concentration was above MIC. Moreover, thepost-antibiotic effect duration was also most closely related to theC_(max)/MIC ratio. Gumbo T, Louie A, Deziel M R, Liu W, Parsons L M,Salfinger M, Drusano G L. Concentration-dependent Mycobacteriumtuberculosis killing and prevention of resistance by rifampin.Antimicrob Agents Chemother. 2007, 51(11):3781-8, the contents of whichare incorporated herein by reference. Thus, to achieve a microbialkilling and to prevent the emergence of resistance in the clinical arenahigh plasma and/or high local concentrations of rifabutin are required.

Emergence of resistance to multiple antimicrobial agents in pathogenicbacteria has become a significant public health threat as there areincreasingly fewer, or even no effective antimicrobial agents availablefor infections caused by these bacteria. Gram-positive and Gram-negativebacteria are both affected by the emergence and rise of antimicrobialresistance.

The life-threatening infections caused by these pathogens are besttreated in hospitals, using optimized dosing regimens that often involveparenteral administration and, in some cases, the additional use ofnebulized antibiotics.

In this context, the intravenous (IV) and inhalation (IN) administrationroutes offer several advantages with respect to oral route in term ofachieving a high cure rate:

a) with the oral route, only a variable fraction reaches the systemiccirculation; the rest of the drug either passes the Gastro-Intestinal(GI) tract without being absorbed, or undergoes first pass effect, i.e.the metabolic transformation occurring in the liver leading to excretionof drug metabolic products through the bile or the kidneys.

b) with the oral route, the C_(max) and the t_(max), the time in whichC_(max) is achieved, are limited by the rate of absorption of the drugin the GI tract.

c) the rate of absorption in humans is very variable depending on thepatient's age, presence of concomitant diseases and the infectiondiseases progression in the patient.

d) with the IV route, instead, the drug enters directly the bloodstream,t_(max) is immediate and C_(max) can be controlled by the drugconcentration infused and the time in which it is infused. AUC, bydefinition, is the maximum that can be achieved with respect to anyother administration route.

e) with the IN route, instead, the drug enters directly the lungs, andC_(max) and distribution in the lungs can be controlled by the drugconcentration and by the particle size produced by the specificnebulizer.

Both IV and IN routes allow rifabutin to reach high AUC/MIC andC_(max)/MIC, which are important for efficacy and protection from drugresistance development during treatment of infections of bacteriasusceptible to the action of rifabutin. The IV route of administrationallow optimization of these parameters in plasma and in any compartmentwhere the drug can properly distribute and is therefore suitable for thetreatment of infections such as e.g., bacteremia, meningitis,Periprosthetic Joint Infections (PJI) and severe lung infections such asVentilator-Associated Bacterial Infections (VABP) and Hospital-AcquiredBacterial Infections (HABP); by IN route is instead possible to achievehigh local concentrations in the lungs for the treatment any bacteriallung infections in all those cases in which a doctor would prefer toachieve very high drug lung concentrations without unnecessarily exposeother body compartments to excessive drug levels. Alternatively, adoctor may decide to use rifabutin by the IN route in combination withan oral or a parenteral antibiotic.

The present invention comprises rifabutin formulations for intravenous(IV) administration. In another embodiment of the invention rifabutin isadministered intravenously (IV) or by inhalation (IN).

Compositions and Formulations Containing Rifabutin

The invention provides compositions that contain formulations in whichfree base rifabutin is dissolved in water/solvent with acid.

The compositions thereof may be provided as pharmaceutically acceptablesalts, such as nontoxic acid addition salts, which are salts of an aminogroup formed with inorganic acids such as but not limited tohydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid andperchloric acid or with organic acids such as, but not limited to,acetic acid, maleic acid, tartaric acid, citric acid, succinic acid,methansulfonic acid, glucuronic acid, malic acid, gluconic acid, lacticacid, aspartic acid, or malonic acid.

In some embodiments, pharmaceutically acceptable salts include, but arenot limited to, adipate, alginate, ascorbate, aspartate,benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate,camphor sulfonate, citrate, cyclopentanepropionate, digluconate,dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate,glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate,hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate,lauryl sulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Preferably, hydrochloric, methanesulfonic, phosphoric, L-tartaric,D-glucuronic, L-malic, D-gluconic, L-lactic, acetic or L-aspartic acidsmay be used. Most preferably, acetic acid, L-lactic acid, D-gluconicacid or D-glucuronic acid is used.

The pharmaceutical composition may be administered by injection,infusion, implantation (intravenous, intramuscular, subcutaneous, or thelike) or by inhalation in dosage forms, formulations, or via suitabledelivery devices or implants containing conventional, non-toxicpharmaceutically acceptable carriers, solvents, diluents, and adjuvants.The formulation and preparation of such compositions are well known tothose skilled in the art of pharmaceutical formulation.

Compositions of the invention for parenteral use may be provided in unitdosage forms (e.g., in single-dose ampoules and vials), in vialscontaining several doses and in which a suitable preservative may beadded (see below), in prefilled syringes, or in prefilled IV bags. Thepharmaceutical compositions described herein may be in the form suitablefor sterile injection. As shown in FIG. 1 , to prepare such acomposition, to rifabutin in powder form, a solution of water in solventwith acid is added to promote dissolution. Alternatively, suchcomposition can be prepared by preparing rifabutin in solvent and mixingit with a solution of water containing the acid.

As such, the invention provides methods of preparing an intravenousformulation of rifabutin. The method may include preparing a solutioncontaining a solvent and distilled water. Preferably, the solution is ina 1:1 ratio or a 1:2 ratio. The solvent may be any solvent, butpreferably is DMI or trascutol HP. An acid may be added to the solution.The acid may be suitable to promote dissolution of rifabutin. The acidmay be any acid, but preferably acetic acid or D-glucuronic acid. Thesolution containing the acid may be added to the rifabutin powder. Thus,the acid causes the rifabutin to dissolve into the aqueous solution.

The rifabutin solution may be added to a pharmaceutically acceptablediluent. The diluent may be 0.9% saline.

For example, to prepare a solution of 20 mg/ml rifabutin in 4% DMI,first a 1:1 solution of dimethyl isosorbide (DMI) in distilled water isprepared. 0.169 ml of glacial acetic acid may then be added to 9.831 mlof the 1:1 DMI/water solution, forming a reconstitution solvent (RS).Then, 1 ml of RS is added to 250 mg of rifabutin to make a solution at250 mg/ml. This RS-rifabutin solution is stirred or shaken until therifabutin dissolves forming a dark deep red solution. Completedissolution should occur in about 15-20 minutes. The concentratedsolution is then diluted with 11.5 ml of water to make a final solutionof rifabutin at 20 mg/ml in 4% DMI. The pH of the final solution isbetween 5 and 6.

In another example to prepare an intravenous solution of 2.5 mg/mlrifabutin in 0.5% DMI/0.9% sodium chloride solution, first a 1:1solution of dimethyl isosorbide in distilled water is prepared. 0.169 mlof glacial acetic acid may then be added to 9.831 ml of the 1:1DMI/water solution forming the RS. Then, 1 ml of RS is added to 250 mgof rifabutin to make a solution at 250 mg/ml. This RS-rifabutin solutionis stirred or shaken until the rifabutin is completely dissolved (about15-20 minutes). The concentrated solution is then diluted with 99 ml of0.9% saline for injection to make a final solution of rifabutin at 2.5mg/ml in 0.5% DMI in 0.9% saline, with a pH between 5.0 and 6.0.

In another example to prepare an intravenous solution of 5 mg/mlrifabutin in 1% transcutol HP/0.9% sodium chloride solution, first a 1:2solution of trancutol HP in distilled water is prepared. 0.169 ml ofglacial acetic acid may then be added to 14.831 ml of the 1:2 transcutolHP/water solution forming the RS. Then, 1.5 ml of RS is added to 250 mgof rifabutin to make a solution at 166.7 mg/ml. This RS-rifabutinsolution is stirred or shaken until the rifabutin is completelydissolved (about 15-20 minutes). The concentrated solution is thendiluted with 48.5 ml of 0.9% saline for injection to make a finalsolution of rifabutin at 5 mg/ml in 1% transcutol HP in 0.9% saline,with a pH between 5.0 and 6.0.

In another example, an intravenous solution of 40 mg/ml rifabutin in 8%DMI/0.9% sodium chloride solution is prepared using a solution of 250 mgof rifabutin in 0.5 ml of DMI, to which 0.5 ml of a solution of 114.6mg/ml of D-glucuronic acid in distilled water is added. After 5 minutesswirling, the resulting RS-rifabutin solution at 250 mg/ml is dilutedwith 5.25 ml of 0.9% saline for injection to make a final solution ofrifabutin at 40 mg/ml in 8% DMI in 0.9% saline, with a pH between 5.0and 6.0.

Depending upon the needs of the patient, and the clinical conditions,administration of the composition by IV administration may be favoredover oral administration because it allows for rapid introduction of theantibiotic into systemic circulation, provides complete bioavailability,allows to better control the pharmacokinetic parameters that are drivingthe pharmacological efficacy, and avoids issues of stability in thegastrointestinal tract and absorption.

The typical dosage of rifabutin is that able to reach plasma or locallevels in which rifabutin C_(max) is >2 mg/L but <50 mg/L and AUC is 10mg*h/L<200 mg*h/L.

The rifabutin solution may be further diluted with a pharmaceuticallyacceptable diluents. For example, a 20 mg/mL IV solution of rifabutinmay be further diluted with 0.9% saline to obtain a lower concentrationof rifabutin for delivery of a less concentrated solution to a subject.Filtration may also be required for IV formulations of rifabutindisclosed herein. If needed, filtration can be performed both on theconcentrated or on the final rifabutin solution.

Formulations of the invention may be for any parenteral administration.For example, the composition may be formulated for injection orinfusion. The injection or infusion may be subcutaneous or intravenous.Preferably, the composition is formulated for intravenousadministration. Preferably, the composition is formulated forintravenous or inhalatory administration. Accordingly, formulations ofthe invention may also include a pharmaceutically acceptable diluent.The pharmaceutically acceptable diluent may be in a concentrationsufficient to deliver a therapeutically effective amount of rifabutin inIV formulation to a patient suffering from an infection. Thepharmaceutically acceptable diluent may be saline or sterile water.Preferably, the diluent is 0.9% saline. The solution may be administeredwith a therapeutically effective amount of rifabutin to treat a patientsuffering from an infection.

It should be noted that the various formulations of the inventiondescribed herein may be used with any of the methods of the invention,and thus the methods are not limited to any singular formulation.

Methods of Preparing Formulations Containing Rifabutin

The invention provides methods for preparing formulations of rifabutin.

FIG. 1 . is a schematic representation illustrating a method forpreparing an injectable rifabutin solution or rifabutin for inhalationaccording to embodiments of the invention. An intravenous rifabutinformulation may be manufactured by a process including preparing asterile pharmaceutically acceptable solution for reconstitutioncomprising a solvent and distilled water in a 1:1 ratio in the presenceof an acid suitable to promote dissolution of rifabutin. Rifabutin maybe present in a solid form or a powder form that is soluble in a liquidmedium. Rifabutin may be dissolved in an aqueous solution of a solventand distilled water. Rifabutin may be soluble in an aqueous solution of50% solvent (i.e., 1:1 solvent-distilled water) in the presence of anacid.

Rifabutin is soluble in an aqueous solution of 33.3% solvent (i.e., 1:2solvent-distilled water) in the presence of an acid.

The formulation may be suitable for any non-oral route ofadministration. The formulation may be suitable for parenteral,intravenous, intraarterial administration. The formulation may besuitable for administration by inhalation.

The formulation may be a reconstituted solution that needs to be dilutedprior to non-oral administration. The formulation may be a reconstitutedsolution suitable for non-oral administration.

The solution may include mixing the solvent and distilled water in adefined ratio. The ratio may be a v/v ratio. The solution may includethe solvent and distilled water in a ratio of from about 9:1 to about1:9, from about 9:1 to about 1:4, from about 9:1 to about 1:2, fromabout 9:1 to about 1:1, from about 4:1 to about 1:9, from about 4:1 toabout 1:4, from about 4:1 to about 1:2, from about 4:1 to about 1:1,from about 2:1 to about 1:9, from about 2:1 to about 1:4, from about 2:1to about 1:2, or from about 2:1 to about 1:1. The solution may includethe solvent and distilled water in ratio of about 9:1, about 4:1, about2:1, about 1:1, about 1:2, about 1:4, or about 1:9.

The solvent may be polyoxyethylene sorbitan monooleate (Tween 80),sorbitan monooleate polyoxyethylene sorbitan monolaurate (Tween 20),polyethylene glycol (PEG), propylene glycol, N-methyl-2-pyrrolidone(NMP), glycerin, ethanol, dimethylacetamide (DMA), diethylene glycolmonoethyl ether (transcutol HP), or dimethyl isosorbide (DMI).

The acid may be hydrochloric, methanesulfonic, phosphoric, L-tartaric,D-glucuronic, L-malic, D-gluconic, L-lactic, acetic, or L-aspartic acid.

The formulation may include diluting the rifabutin-containing solutioninto a diluent. The diluent may be sterile water, sodium chloride (i.e.,saline) solution, dextrose water, or Ringer lactate solution. The sodiumchloride solution may be a 0.9% sodium chloride solution. The dextrosesolution may be a 5% dextrose solution or a 10% dextrose solution.

The obtained solutions may require further dilution in apharmaceutically acceptable solvent such as including but not limitedto, sterile water, mannitol, such as 3-5% mannitol, 3% mannitol, 4%mannitol, 4.3% mannitol, and 5% mannitol, phosphate, acetate, additionaltartrate, saline, such as physiological saline (0.9%), ½ physiologicalsaline (0.45%), and 0.5% saline, and the like. For intravenousformulations, physiological saline (0.9%) is a preferred diluent orcarrier.

Rifabutin may be reconstituted in a solution containing an acid in aquantity comprised between 1 and 3 molar equivalents in a solvent andwater mixture in ratios v/v of from about 9:1 to about 1:9. Therifabutin to solvent ratio in w/v may be from 4:1 to 1:4. Thedissolution times for obtaining the formulations may be less than 60minutes. In particular the amount of acid may be 1 molar equivalent, theacid may be acetic acid or D-glucuronic acid, the solvent may be DMI ortranscutol HP mixed with water in a 1:1 or 1:2 v/v ratio, the rifabutinto solvent ratio w/v may be 1:2 and the dissolution time is less than 20minutes.

The formulation (i.e., the reconstituted rifabutin solution) may be usedas such or may be made by diluting it into a defined volume of diluent.The volume of diluent may be expressed relative to the volume ofreconstituted solution. The volume of diluent may be from about 1.0 toabout 2.0 volumes, from about 1.25 to about 2.25 volumes, from about 1.5to about 2.5 volumes, from about 1.75 to about 2.75 volumes, from about2.0 to about 3.0 volumes, from about 1.80 to about 2.10 volumes, fromabout 1.90 to about 2.05 volumes, or from about 1.95 to about 2.0volumes of the reconstituted rifabutin solution. The reconstitutedrifabutin solution may be added to from about 20.5 to about 30 volumes,from about 21 to about 29 volumes, from about 22 to about 28 volumes,from about 23 to about 27 volumes, from about 23 to about 26 volumes,from about 23 to about 25 volumes, from about 23 to about 24 volumes,from about 22.5 to about 23.5 volumes, or from about 23.0 to about 23.5volumes of 0.9% saline for injection. Accordingly, one volume of diluentleads to a final solution of about 125 mg/ml, 4 volumes leads to a finalsolution of about 50 mg/ml, 9 volumes leads to a final solution of about25 mg/ml, 24 volumes leads to a final solution of about 10 mg/ml, and 99volumes leads to a final solution of about 2.5 mg/ml. The skilledartisan understands how to make a solution with any desired finalconcentration.

The formulation may contain rifabutin at any suitable concentration,such as those described above.

The formulation may contain DMI at any concentration, such as thosederiving from the dilutions described above.

The formulation may contain transcutol HP at any concentration, such asthose deriving from the dilutions described.

The amount of acid relative to rifabutin may be between 1 and 3 molarequivalents or between 1 and 2 molar equivalents. The amount of acidrelative to rifabutin may be 1 molar equivalent.

The w/v ratio of rifabutin to solvent may be from about 4:1 to about1:4, from about 2:1 to about 1:3, or from about 1:1 to about 1:2. Thew/v ratio of rifabutin to solvent may be about 4:1, about 3:1, about2:1, about 1:1, about 1:2, about 1:3, or about 1:4.

The method may include dissolving the rifabutin by swirling, stirring,or agitation of the solution. The dissolving step may be performed for adefined period. The dissolving step may be performed for about 5minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30minutes, about 45 minutes, or about 60 minutes.

Methods of Treating Bacterial Infections

The invention provides methods of treating a bacterial infection. Themethods include administering a liquid formulation of rifabutin to asubject with a bacterial infection. The liquid formulation may includerifabutin, a solvent and an acid.

The formulation may be provided by intravenous, intraarterial, orpulmonary administration. The formulation may be provided by inhalationor by injection.

The liquid formulation may be a solution of rifabutin and a diluent tobe administered intravenously to a subject with a bacterial infection.The formulation for IV administration may include a pharmaceuticallyacceptable solvent. The method may include administering an IVformulation of any formulation of rifabutin described herein to asubject suffering from a bacterial infection.

The IV formulation containing rifabutin may be administered with anotherantibiotic or therapeutic. Sequential administration or alternatingadministration may include providing IV formulation containing rifabutinexclusively for a period of time and providing the other therapeuticexclusively for a period of time. Sequential administration may includea period of overlap in which the subject is provided both the IVformulation containing rifabutin and the formulation containing theother therapeutic. The periods of exclusivity and periods of overlap mayindependently be 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 1day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 10months, 12 months, 18 months, or 24 months. Alternatively, thepharmaceutical formulations of the invention, or the soluble componentswithin the formulation, may contain rifabutin and another therapeutic.

Without wishing to be bound to theory, any formulations of the inventionmay be used in any of the methods of the invention.

In another aspect of the invention, methods include treating a bacterialinfection in a subject. The method may include administering atherapeutically effective amount of a formulation comprising rifabutinor a salt thereof. The rifabutin formulation may be formulated forintravenous administration. The intravenous formulation is manufacturedby a process comprising preparing a solution of a solvent and distilledwater in a 1:1 ratio in the presence of an acid suitable to promotedissolution of said rifabutin. Preferably, the solvent is DMI.Preferably, the solvent is transcutol HP. Preferably the intravenousformulation is approximately 2.5 mg/mL rifabutin in 0.5% DMI and 0.9%sodium chloride solution.

In another embodiment, the method may include providing a subjectsuffering from a bacterial infection a combination therapy of an IVformulation of rifabutin or a salt thereof and another therapeutic. TheIV formulation may include a pharmaceutically acceptable solvent. Thetherapeutic may be in a formulation for IV administration.

The IV formulation containing rifabutin and the formulation containinganother therapeutic may be provided or administered simultaneously,sequentially in either order, or in an alternating manner. Sequentialadministration or alternating administration may include providing IVformulation containing rifabutin exclusively for a period of time andproviding the formulation containing the other therapeutic exclusivelyfor a period of time. Sequential administration may include a period ofoverlap in which the subject is provided both the IV formulationcontaining rifabutin and the formulation containing the othertherapeutic. The periods of exclusivity and periods of overlap mayindependently be 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 1day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 10months, 12 months, 18 months, or 24 months.

In another embodiment rifabutin is filled into glass vials andsterilized using one of the procedures used for terminal sterilization.Such procedures are well known to those skilled in the art and may beperformed using gamma radiation or thermal sterilization.

A 50:50 sterile solution of water for injection and a solvent containingapproximately 1 molar equivalent of an acid is then added to sterilerifabutin to form the concentrated reconstituted solution.

In another embodiment the reconstituted rifabutin formulation isfiltered sterile.

In another embodiment the diluted rifabutin formulation is filteredsterile.

EXAMPLES Example 1

Preliminary assessment of solubility in solvents: To determine ifpharmaceutical acceptable solvents could dissolve rifabutin powder andat which concentrations, an adequate volume of solvent sufficient toachieve a maximum target solubility of 300 mg/ml was added to smallaliquots of rifabutin powder. After 24 h stirring at room temperature,samples were centrifuged, and the supernatant analyzed by HPLC. Therifabutin chromatographic peak area was compared against a titrationcurve. Results of this preliminary screening are reported in Table 1.

TABLE 1 Solubility results in formulation vehicles Solvent/solutionSolubility (mg/ml) at 24 h Polyethylene glycol 400 (PEG400) ≥110Propylene glycol ≥92 N-Methyl-2-pyrrolidone (NMP) ≥190 Dimethylacetamide(DMA) ≥204 Transcutol HP ≥206 Glycerol 0.4 Tween 80 (1% w/w in water)1.3 Povidone K12 (5% w/w in water) 0.5 Dimethyl isosorbide (DMI) ≥245Saline 0.9% 0.3 Acetate buffer pH 4 (USP) 11.3 Phosphate buffer pH 6(USP) 0.3 Phosphate buffer pH 8 (USP) 0.2 Borate buffer pH 10 (USP) 1.9Ethanol (literature data) 30

The “thermodynamic” solubility screen showed dimethyl isosorbide (DMI)as the best solvent. Although interesting, solubility data at 24 h areirrelevant in a reconstitution process of an antibiotic powder since asolvent for reconstitution must be able to dissolve the antibiotic in afew minutes, a time that is practical for an operator to preparerifabutin for injection or for inhalation.

Dissolution of rifabutin in DMI at concentrations of >250 mg/ml waspossible, but proven to be longer than plausibly acceptable. Inaddition, dilution of the DMI rifabutin solution with pharmaceuticallyacceptable solutions such as saline was impossible since theDMI-rifabutin solution separated from the aqueous solution.

Unexpectedly, addition of acid to a DMI/water solution allowed a rapiddissolution of rifabutin powder and, the highly concentratedreconstituted rifabutin solution could be diluted ad libitum with wateror 0.9% saline without any rifabutin precipitation.

Example 2

Small scale solubility screen of solvents and acids: an amount ofrifabutin powder comprised between 100 and 200 mg were weighed into 1.8ml glass vials. Examples of solutions for reconstitution were preparedby mixing different solvents and water containing different acids in theamounts as specified in Table 2. The solutions were stirred at RT for 15minutes using a magnetic stirring bar and were briefly vortexed. Thesamples were filtrated using centrifugal filters (0.2 μm PTFE filter),and the concentration the reconstituted solutions was determined by HPLCanalysis. The rifabutin chromatographic peak area was compared against atitration curve and results are reported in Table 2.

TABLE 2 Small scale solubility screen RBT/ RBT/acid Solvent/ Time toApprox. Example solvent ratio water dissolution conc. No. Solvent Acidratio (w/v) (mole/mole) ratio (v/v) (min) Notes (mg/ml) 1 DMI — 1:2 —1:1 ≥30 not dissol. 86 2 DMI HCl 1:2 1:1 1:1 ca. 20 dissolved 290 3 DMIMethanesulfonic 1:2 1:1 1:1 ca. 20 dissolved 280 4 DMI Phosphoric 1:21:1 1:1 ca. 20 dissolved 300 5 DMI L-tartaric 1:2 1:1 1:1 ca. 20dissolved 240 6 DMI D-glucuronic 1:2 1:1 1:1 ca. 20 dissolved 300 7 DMIL-malic 1:2 1:1 1:1 ca. 20 dissolved 300 8 DMI D-gluconic 1:2 1:1 1:1ca. 20 dissolved 300 9 DMI L-lactic 1:2 1:1 1:1 ca. 20 dissolved 260 10DMI acetic 1:2 1:1 1:1 ca. 20 dissolved 290 11 DMI aspartic 1:2 1:1 1:1≥30 not dissol. 40 12 DMA D-glucuronic 1:2 1:1 1:1 ca. 20 dissolved ≥25013 DMA acetic 1:2 1:1 1:1 ca. 20 dissolved ≥250 14 PEG400 — 1:2 — 1:1≥30 not dissol. not done 15 PEG400 D-glucuronic 1:2 1:1 1:1 ≥30 notdissol. not done 16 PEG400 acetic 1:2 1:1 1:1 ≥30 not dissol. not done17 Transcutol D-glucuronic 1:2 1:1 1:2 ca. 15 dissolved 130 18Transcutol acetic 1:2 1:1 1:2 ca. 15 dissolved — 19 DMI D-glucuronic 1:21:1 1:2 ca. 15 dissolved 150 20 DMI acetic 1:2 1:1 1:2 ca. 15 dissolved160

TABLE 3 Solubility of rifabutin reconstituted solutions at t = 0 and t =24 h Solubility Solubility Solubility Example (mg/ml) (mg/ml) (mg/ml)No. Solvent/water mix Acid T0 T 24 h RT T 24 h 5° C. 2 DMI/water 1:1Hydrochloric 290 250 280 3 DMI/water 1:1 Methanesulfonic 280 280 300 4DMI/water 1:1 Phosphoric 300 210 190 5 DMI/water 1:1 L-Tartaric 240 230250 6 DMI/water 1:1 D-Glucuronic 300 260 260 7 DMI/water 1:1 L-Malic 300200 210 8 DMI/water 1:1 D-Gluconic 300 260 260 9 DMI/water 1:1 L-Lactic260 200 290 10 DMI/water 1:1 Acetic 290 250 270 11 DMI/water 1:1L-Aspartic  40  40  40 17 Transcutol/water 1:2 D-glucuronic 130 172 — 18Transcutol/water 1:2 acetic — 130 — 19 DMI/water 1:2 D-glucuronic 150180 — 20 DMI/water 1:2 acetic 160 150 —

TABLE 4 Solubility and pH at t = 0 and t = 24 h of reconstitutedrifabutin solutions diluted with 0.9% saline Dilution SolubilitySolubility Example in 0.9% (mg/mL) (mg/mL) pH pH No. Solvent/water mix +Acid saline T 0 T 24 h RT T 0 h T 24 h 1 DMI/water 1:1 + — 10x 2 2 7.16.6 2 DMI/water 1:1 + Hydrochloric 10x 23 18 2.5 2.6 3 DMI/water 1:1 +Methanesulfonic 10x 25 28 2.5 2.6 4 DMI/water 1:1 + Phosphoric 10x 20 242.5 2.6 5 DMI/water 1:1 + L-Tartaric 10x 23 23 3.2 3.2 6 DMI/water 1:1 +D-Glucuronic 10x 26 28 3.8 4.0 7 DMI/water 1:1 + L-Malic 10x 19 22 3.63.6 8 DMI/water 1:1 + D-Gluconic 10x 16 25 5.8 4.3 9 DMI/water 1:1 +L-Lactic 10x 20 25 4.1 4.2 10 DMI/water 1:1 + Acetic 10x 26 25 5.1 5.211 DMI/water 1:1 + L-Aspartic 10x 3 3 6.3 6.1 17 Transcutol/water 1:2 +D-glucuronic    6.67x 22 23 — — 18 Transcutol/water 1:2 + acetic   6.67x 21 22 — — 19 DMI/water 1:2 + D-glucuronic    6.67x 22 24 — — 20DMI/water 1:2 + acetic    6.67x 19 18 — — 1 DMI/water 1:1 + — 100x  0.30.4 7.2 7.3 2 DMI/water 1:1 + Hydrochloric 100x  2.5 2.5 4.2 3.9 3DMI/water 1:1 + Methanesulfonic 100x  2.6 2.6 4.1 3.8 4 DMI/water 1:1 +Phosphoric 100x  2.3 2.2 3.5 3.3 5 DMI/water 1:1 + L-Tartaric 100x  2.32.2 3.4 3.4 6 DMI/water 1:1 + D-Glucuronic 100x  2.5 2.3 4.8 4.2 7DMI/water 1:1 + L-Malic 100x  1.9 1.9 3.9 3.7 8 DMI/water 1:1 +D-Gluconic 100x  2.2 1.9 5.9 4.4 9 DMI/water 1:1 + L-Lactic 100x  2.52.4 4.5 4.3 10 DMI/water 1:1 + Acetic 100x  2.5 2.3 5.2 5.2 11 DMI/water1:1 + L-Aspartic 100x  0.4 0.4 6.6 6.5

Example 3

Large scale evaluation for reconstitution of rifabutin and furtherdilution with 0.9% saline: 1.50 g (1.77 mmoles) of rifabutin powder wereweighed into a 40 mL glass vial. An equimolar amount of acid (1.77mmoles) dissolved in 6 mL dimethyl isosorbide/water 50/50 (v/v) wasadded. The solutions were vortexed for 30 seconds, vigorously stirred atRT for 15 minutes using a magnetic stirring bar and again vortexed for30 seconds.

The samples were filtrated, using a syringe and filter (0.2 μm PTFEfilter), and the concentration of reconstituted rifabutin solution wasdetermined by HPLC analysis. Aliquots (1 mL) of the undiluted solutionswere stored at RT and at 5° C. for 24 hours.

FIG. 2 is a schematic representation illustrating methods of analysis ofrifabutin formulations. Aliquots (500 μL) of the filtrate were diluted10-fold and 100-fold in 0.9% saline. These dilutions were prepared intriplicate. The diluted samples were visually inspected for immediateprecipitation and were filtered after 15 min. The concentration of APIwas determined by HPLC analysis and the pH was recorded. The dilutedsamples were stored at RT for 24 hours.

After the storage time of 24 hours, the diluted and undiluted sampleswere reanalyzed by HPLC analysis for determination of the APIconcentration and the pH was recorded.

The experimental details, results and recorded pH values of thesolubility tests of the undiluted samples are shown in Tables 5, 6 and 7and graphically represented in FIGS. 3 and 4 .

TABLE 5 Concentration and pH of reconstituted rifabutin solutions at t =0 and stored for 24 hours at 5° C. and room temperature Time = 24 hoursTime = 24 hours Time = 0 Room temperature 5° C. ConcentrationConcentration Concentration Acid mg/ml pH mg/ml pH mg/ml pH Acetic acid244 6.3 257 6.2 262 6.3 L-Lactic acid 263 5.9 270 6.2 236 6.2 D-Gluconicacid 224 6.3 211 5.9 216 5.9 D-Glucuronic acid 241 5.9 241 5.7 262 5.8

FIG. 3 is a graph showing rifabutin solubility in formulations.Undiluted samples at 5 larger scale were analyzed at to (blue bars),after 24 hours at room temperature (red bars), and after 24 hours at 5°C. (green bars).

TABLE 6 Concentration and pH of rifabutin solutions at a nominalconcentration of 25 mg/ml stored for 24 hours at room temperature*.reconstituted 10 fold dilution 10 fold dilution solution Time = 0 Time =24 hours Concentration Concentration Concentration Acid mg/ml pH mg/mlpH mg/ml pH Acetic acid 244 6.3 22.3 ± 3.4 5.7 25.3 ± 2.4 5.6 L-Lacticacid 263 5.9 24.6 ± 1.2 6.5 21.6 ± 0.7 5.4 D-Gluconic acid 224 6.3 23.5± 1.0 6.6 22.5 ± 1.6 4.5 D-Glucuronic acid 241 5.9 25.6 ± 0.6 5.7 26.3 ±1.2 5.6 *The experiment has been performed in triplicate.

Osmolality of these solutions was 632, 644, 622 and 645 mOsm/kg,respectively for the solutions generated from acetic acid, L-lacticacid, D-gluconic acid and D-glucuronic acid.

TABLE 7 Concentration and pH of rifabutin solutions at a nominalconcentration of 2.5 mg/ml stored for 24 hours at room temperature*.reconstituted 100 fold dilution 100 fold dilution solution Time = 0 Time= 24 hours Concentration Concentration Concentration Acid mg/ml pH mg/mlpH mg/ml pH Acetic acid 244 6.3 2.4 ± 0.0 5.6 2.4 ± 0.0 5.6 L-Lacticacid 263 5.9 2.3 ± 0.0 5.9 2.2 ± 0.0 5.7 D-Gluconic acid 224 6.3 2.0 ±0.0 6.0 2.0 ± 0.0 4.5 D-Glucuronic acid 241 5.9 2.3 ± 0.0 5.6 2.3 ± 0.05.4 *The experiment has been performed in triplicate.

Osmolality of these solutions was 323, 319, 316 and 318 mOsm/kg,respectively for the solutions generated from acetic acid, L-lacticacid, D-gluconic acid and D-glucuronic acid.

FIG. 4 is a graph showing rifabutin solubility in formulations.

Diluted samples were analyzed at to (blue bars, 10× dilution; grey bars100× dilution) and after 24 hours at room temperature (orange bars, 10×dilution; yellow bars 100× dilution).

Example 4

Reconstitution of rifabutin from a solution of rifabutin in transcutolHP and further dilution with 0.9% saline: 1.50 g (1.77 mmoles) ofrifabutin powder were weighed into a 40 mL glass vial. 3 ml of DMI or 3ml of transcutol HP were added and the very thick suspension was swirledfor about 6 and 18 hours, respectively in order to obtain a very densesolution. The solutions were filtered sterile through a 0.2 μm filter(PTFE filter). To an aliquot of 1 ml of the two solutions containingabout 500 mg (about 0.590 mmols) of rifabutin, 1 ml of a solution ofwater containing 0.590 mmols of acetic acid was added under gentleswirling. A perfect solution formed extemporarily. Analysis pre and postfiltration (0.2 μm PTFE filter) show a similar titer for all solutionsin agreement with the results reported in Example 3.

To another aliquot of 1 ml of the two solutions containing about 500 mg(about 0.590 mmols) of rifabutin, 5 ml of a solution of water/0.9%saline 1:4 containing 0.590 mmols of acetic acid were added. A perfectsolution formed extemporarily. Analysis pre and post filtration (0.2 μmPTFE filter) show a similar titer of all solutions from about 46.7 to48.2 mg/ml.

Example 5

Analytical methods and sample preparation for the determination of thesolubility: Dilutions for the solubility determination were prepared byadding 25 μL of the mother liquor to 500 μL of 0.1% TFA in acetonitrile(dilution 1 21-fold). If necessary, a second dilution was prepared byadding 25 μL of dilution 1 to 500 μL of 0.1% TFA in acetonitrile(441-fold dilution).

LCMS Method HPLC: Agilent 1200 Detector 1: DAD set at 276 nm Detector 2:Mass Spectrometer HPLC Conditions:

Column: Sunfire C18 (100×4.6 mm×3.5 μm)Column temp: 35° C.Flow cell: 10 mm pathMobile phase A: 0.1% TFA in WaterMobile phase B: 0.1% TFA in AcetonitrileFlow: 1.0 ml/min

HPLC mobile Mobile Mobile phase gradient. phase A phase Time (min) (%) B(%) 0 90 10 9 10 90 10 90 10 11 90 10

Rifabutin had a retention time of 6.8-6.9 min. The peak area observedfrom the UV detector of the compound of interest was employed for thecalculation of concentration of the component in the solution. It wasverified that there was no interference with the ingredients of thevehicles.

Table 3 and Table 5 report the concentration of rifabutin in solutionscontaining 1 molar equivalent of an acid in dimethyl isosorbide/water50/50 (v/v) or transcutol HP/water 33.3/66.7 (v/v) freshly prepared andafter storage at room temperature (RT) or 5° C. for 24 hours.

Reconstituted solutions can be stored for 24 hours at RT and at 5° C.

Table 4, Table 6 and Table 7 report the concentration of rifabutin afterup to 100-fold dilution in 0.9% saline and storage at RT for 24 hours.The pH and osmolitity of the solutions was also recorded.

Reconstituted solutions may be diluted without restriction in order torender a composition suitable for a desired route of administration.

Dilution of reconstituted solutions with 0.9% saline has a final pH thatdepends on the pKa of the acid used, and a preferred acid should have apKa value greater than 2, preferably greater than 3. Preferably suchacids are D-glucuronic acid, D-gluconic acid, L-lactic acid and aceticacid. Most preferably the acid is acetic acid or D-glucuronic acid.

Example 6

Reconstitution of Rifabutin from a Large Scale Solution of Rifabutin inDMI and Further Dilution with 0.9% Saline:

Preparation of vial 1: 1200 ml of DMI was heated at 40° C. in a 5 Lglass tank and 600 g (0.708 moles) of rifabutin powder is addedportion-wise under stirring at 40° C. Complete dissolution was obtainedin about 6 hours and the solution was allowed to return to roomtemperature. The solution was then filtered through a PVDF(polyvinylidene fluoride), Nylon, or PTFE (polytetrafluoroethylene) 0.22μm sterile filter. A volume of solution equivalent to 500 mg (0.590mmoles) of rifabutin was transferred into 10 ml sterile anddepyrogenized vials via sterile tubing and the vials were sealed withFluorotec rubber stoppers and flip-off overseals. Additionally oralternatively, the vials underwent terminal sterilization at 121° C. for20 minutes in an autoclave. This procedure was repeated starting fromtwo different rifabutin batches.

Analysis and stability data after terminal sterilization are reported inTables 8 and 9, respectively.

Preparation of vial 2: a solution of 4% w/v acetic acid in sterile waterfor injection was transferred into 10 ml sterile and depyrogenized vialsvia sterile tubing and the vials were sealed with Fluorotec rubberstoppers and flip-off overseals and underwent terminal sterilization inan autoclave.Reconstitution of rifabutin before use: 1 ml of sterile 4% w/v aceticacid (0.66 mmoles) in water for injection was withdrawn by means of 1 mlsyringe from vial 2 and was added to vial 1 under gentle swirling.Dilution of reconstituted rifabutin solution in 0.9% saline: Thereconstituted solution was brought to a total volume of 10 ml byaddition of sterile solution of 0.9% NaCl solution (saline forinjection) to bring final concentration of rifabutin to 50 mg/ml.Analysis data are reported in Table 10Alternatively, the reconstituted solution can be withdrawn from the vialby means of a syringe and directly injected into a saline bag forinfusion.

TABLE 8 Analysis of vial 1 prepared from two different rifabutin batchesCompliance and results From API From API Test batch 1 batch 2 Appearance(vial crimped and sealed) comply comply Color and clarity(reddish-violet dense comply comply solution, free of visible particles)Vial content in mg/ml (500 mg ± 10%)   507.6   492.2 Related Impurity E   0.0 *    0.0 * substances Impurity B  −0.1 *    0.1 * Impurity D   0.0 *    0.0 * Impurity C    0.0 *  −0.1 * RRT1.6-1.7  −0.05;   0.0 *  −0.1 *   Any other impurity    0.3 *    0.0 * Total of allimpurities    1.1 *    0.1 * *Variation in impurity content of rifabutinin vial l from rifabutin drug substance (API). Data are impurity % andare reported as % impurity in vial 1-% impurity in API.

TABLE 9 Stability results of vial 1 stored at 2-8° C. and at 25° C. ± 2°C./60% RH ± 5% RH Stability results * Stability results * Vial 1 storedat 2-8° C. Vial 1 stored at 25° C./60% RH T = 15 T = 30 T = 90 T = 15 T= 30 T = 90 Test days days days days days days Related Impurity E 0.10.1 0.0 0.1 0.1 0.0 substances Impurity B −0.2 −0.2 −0.2 −0.2 −0.2 −0.2Impurity D 0.0 0.0 0.0 0.0 0.0 0.0 Impurity C 0.0 0.0 0.0 0.0 0.0 0.0RRT1.6-1.7 0.0 0.0 0.05; 0.1 0.0 0.0 0.25; 0.3 Any other 0.1 0.0 0.0 0.10.1 −0.1 impurity Total of all 0.3 0.3 −0.1 0.4 0.4 −0.1 impurities *Variation in impurity content of rifabutin in vial 1 upon storage atdifferent conditions. Data are impurity % and are reported as % impurityin vial 1 at day 15, 30 and 90 - % impurity in vial 1 at day 0.

TABLE 10 Analysis of reconstituted solutions after dilution to 50 mg/ml0.9% saline Test Compliance and results Color and clarity(reddish-violet dense comply solution, free of visible particles)   51.7Rifabutin concentration in mg/ml (50 mg/ml ± 10%) pH    5.6 RelatedImpurity E   substances Impurity B  −0.1 * Impurity D    0.0 * ImpurityC  −0.05 * RRT1.6-1.7    0.15;    0.2 * Any other impurity  −0.3 * Totalof all impurities  −0.6 * * Variation in impurity content of rifabutinafter reconstitution and dilution with 0.9% saline Data are impurity %and are reported as % impurity in reconstituted/diluted solution-%impurity in vial l.

Example 7

Analytical Methods and Sample Preparation for Stability Studies andImpurity Determination

HPLC Conditions

HPLC HPLC Waters Alliance equipped with UV detector or Apparatusequivalent Software Empower 3 System or equivalent Column C8, 5 μm, 4.5× 150 mm, Waters Spherisorb Flow rate 1.0 ml/min Injection volume 10 μlWavelength 254 nm Mobile phase 55% ACN + 45% of 13.6 g/L (0.1M) ofpotassium dihydrogen phosphate. The mixture has to be adjusted to pH6.5± 0.1 with 2N NaOH Elution Isocratic Run time 2.5 times the retentiontime of rifabutin RRT Rifabutin: 1 (about 9 min) (with reference to Imp.E: about 0.5 Rifabutin) Imp. B: about 0.6 Imp. D: about 0.8 Imp. C:about 1.4

Sample Preparation

Blank solution: mobile phase as it is.

Test Solution 1—for Determination of the Concentration of the Rifabutinin a Bulk Solution:

0.5 mg of Rifabutin was transferred to a solution in DMI, accuratelyweighed, to a 10-mL volumetric flask and diluted to volume withacetonitrile. 1.5 ml of the obtained solution was transferred into a 50ml-volumetric flask, dilute with mobile phase to volume, and mixed (0.5mg/ml).

Test Solution 2—for the Vial Content Determination:

After removal of the vial flip-off, about 5 ml of ACN in the sealed vialwas transferred by using a 10 ml syringe. The obtained solution was thentransferred into a 50 ml volumetric flask and washed at least 5 timeswith ACN by adding the washing solution into the 50 ml volumetric flask,in order to obtain an accurate recovery of the whole amount of solutionof the vial. The seal and the stopper was then removed and another twowashes of the vial were performed. The volume was then diluted with ACN.

Lastly the obtained solution was diluted 1 ml to 20 ml with the mobilephase and mix (0.5 mg/ml).

-   Standard solution: about 25 mg of Rifabutin CRS, accurately weighed,    was transferred to a 50-mL volumetric flask. 5 mL of acetonitrile    was added and the solution was diluted with mobile phase to volume,    and mixed (0.5 mg/ml).-   Diluted Standard solution: 1 ml of Rifabutin standard solution was    diluted to 100 ml with mobile phase (0.005 mg/ml).-   Resolution solution: about 10 mg of Rifabutin CRS was dissolved in 2    ml MeOH, 1 ml of 2N NaOH was added and the solution allowed to stand    for about 4 min. 1 ml of 2N HCl was added and the solution diluted    to 50 ml with the mobile phase.

System Suitability

-   -   On the Resolution solution: the chromatogram exhibited a major        peak for a degradant, two minor peaks for degradants, and a        major peak for Rifabutin at RRT of about 0.5, 0.6, 0.8, and 1.0,        respectively. The resolution between the Rifabutin peak and the        degradant peak eluting at a relative retention time of about 0.8        was not less than 1.3.    -   On the Standard preparation: the column efficiency was not less        than 2000 theoretical plates, and the relative standard        deviation for replicate injections was not more than 2.0%.

Calculations for Test Solution 1

Calculation of the quantity, in mg, of Rifabutin in each g of sample isperformed using the formula:

${{mg}{Rifabutin}} = {\left( \frac{C \times P}{W} \right) \times \left( \frac{AT}{AS} \right) \times \frac{50 \times 10}{1.5}}$

-   -   in which

C=standard concentration (mg/mL)

P=standard potency (μg/mg),

W=sample weight (g)

AT=area of sample peak

AS=average area of standard peak

Calculation of the percentage of each impurity is performed using theformula:

${\%{each}{impurity}} = {\left( \frac{C \times P}{W} \right) \times \left( \frac{AI}{AS} \right) \times \frac{50 \times 10}{1.5 \times R}}$

-   -   in which

C=diluted standard concentration (mg/mL)

P=standard potency (μg/mg)

W=sample weight (g)

AI=area of impurity peak

AS=area of standard peak

R=mg Rifabutin in each g of sample calculated concentration (mg/g)

Calculations for Test Solution 2

Calculation of the content of Rifabutin, in g, in each vial is performedusing the formula:

${{mg}{Rifabutin}{per}{vial}} = {\left( {C \times P} \right) \times \left( \frac{AT}{AS} \right) \times \frac{50 \times 20}{1}}$

-   -   in which

C=standard concentration (mg/mL)

P=standard potency (μg/mg),

W=sample weight (g)

AT=area of sample peak

AS=average area of standard peak

Calculation of the percentage of each impurity is performed using theformula:

${\%{each}{impurity}} = {\left( {C \times P} \right) \times \left( \frac{AI}{AS} \right) \times \frac{50 \times 20}{1 \times R}}$

-   -   in which

C=diluted standard concentration (mg/mL)

P=standard potency (μg/mg)

W=sample weight (g)

AI=area of impurity peak

AS=area of standard peak

R=mg Rifabutin in each vial

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification, and guidance that can be adapted to the practice ofthis invention in its various embodiments and equivalents thereof.

1. A rifabutin formulation produced from a rifabutin powder in thepresence of an acid, water, and a solvent suitable to promotedissolution of the rifabutin.
 2. The formulation of claim 1, wherein theratio of rifabutin to solvent w/v is about 1:2.
 3. The formulation ofclaim 1, wherein the solvent is selected from the group consisting of:PEG, propylene glycol, NMP, ethanol, DMA, transcutol HP, and dimethylisosorbide (DMI).
 4. The formulation of claim 3, wherein the solvent isDMI or trascutol HP.
 5. The formulation of claim 1, wherein the solventto water ratio v/v is from about 1:1 to about 1:2.
 6. The formulation ofclaim 1, wherein the acid is selected from the group consisting of:hydrochloric, methanesulfonic, phosphoric, L-tartaric, D-glucuronic,L-malic, D-gluconic, L-lactic, acetic and L-aspartic.
 7. The formulationof claim 6, wherein the acid is acetic acid or D-glucuronic acid.
 8. Theformulation of claim 1, wherein the rifabutin to acid molar ratio isabout 1:1.
 9. The formulation of claim 1, wherein the formulation isdiluted in order to produce a composition suitable for a desired routeof administration.
 10. A method of preparing a formulation of rifabutin,the method comprising: preparing a solution comprising a solvent, waterand an acid; and adding said solution to rifabutin powder, therebycausing the rifabutin to dissolve in the solution.
 11. The method ofclaim 10, wherein the solvent is selected from DMI and tanscutol HP. 12.The method of claim 10, wherein the acid is selected from acetic acidand D-glucuronic acid.
 13. A method of preparing a formulation ofrifabutin, the method comprising: preparing a solution comprising waterand an acid; and adding said solution to a rifabutin solution in asolvent, thereby producing an aqueous rifabutin formulation.
 14. Themethod of claim 13, wherein the solvent is selected from DMI andtanscutol HP.
 15. The method of claim 13, wherein the acid is selectedfrom acetic acid or D-glucuronic acid.
 16. (canceled)